The MLL gene at chromosome band 11q23 is rearranged frequently in de novo and in therapy-related leukemia. The consequence of these translocations is the formation of a chimeric gene that consists of N-terminal sequences from MLL fused in frame to C-terminal sequences of its partner genes. Previously, we have cloned the ELL gene involved in the (11;19)(q23; p 13.1) translocation, one of the most common of 11q23 aberrations. To characterize these leukemias in detail, we have used two alternative strategies to develop mouse models of MLL-ELL leukemia. Using retroviral infection of bone marrow followed by transplantation, recipient mice all develop acute leukemia in 4-6 months. In contrast, in a knock-in model of MLL-ELL generated by gene targeting, chimeric and heterozygous mice do not develop leukemia and exhibit no apparent hematopoietic phenotype, indicating that expression of the MLL-ELL fusion protein is insufficient by itself for the development of acute leukemia. However, treatment of the knock-in mice with ENU induces acute leukemia in 90% of mice that recapitulates the phenotype observed in the retroviral transplant model and human acute leukemias in patients with (11; 19)(q23;p13.1) translocations. Recent analyses of blood samples obtained at birth from childhood leukemia patients have revealed that secondary genetic events are also required in the development of human leukemias. These data indicate that "second hits" are necessary for the development of MLL-ELL leukemia. Using the MLL-ELL knock-in model, we propose to identify the cooperating mutations that are required for the development of MLL leukemia. To determine the critical functional requirements of MLL fusion proteins and to elucidate the nature of the breakpoint cluster region of MLL, we plan to use a hematopoietic immortalization assay to define the domains of MLL that must either be included or excluded from MLL fusion proteins. The development of these models will provide critical reagents for the characterization of 11q23 leukemias, the analysis of the contributions of individual domains within the MLL fusion genes to leukemogenesis, and the development of novel treatment strategies for this common subtype of leukemia.